1. Field of the Invention
The present invention relates to a protein-free culture medium useful for the routine large-scale production of monoclonal antibodies and other cell-produced substances of biological and clinical importance.
2. Description of the Prior Art
The use of antibodies in all areas of medicine and biology has expanded rapidly since the 1975 publication by Kohler and Milstein of their technique which allows the production of monoclonal antibodies of desired specificity. Previously used conventional antisera, sometimes known as polyclonal antibodies, were reactive with many determinants and thus were of limited use in identifying or targeting specific determinants. In contrast, monoclonal antibodies possess homogeneity, thus greatly facilitating the standardization and reproducibility of immunochemical procedures. However, it is necessary to obtain the antibodies in a highly purified state if many of their desirable properties are to be fully realized, especially if they are to be used therapeutically.
Currently, monoclonal antibodies are generally produced by growing cells in the presence of serum, either in vivo by ascites tumors or by cultivation in vitro in a medium supplemented with serum, preferably antibody-free serum. Since both ascites fluid and serum-supplemented culture media are highly complex mixtures of proteins and other molecules, and since the monoclonal antibodies being produced are themselves proteins, purification of the antibodies from the mixture is difficult and time-consuming. Furthermore, so-called antibody-free serum such as fetal calf serum (which is, in fact, never completely free of antibody) is extremely expensive and is a significant factor in determining the overall cost of the media.
Accordingly, previously investigators have attempted to grow hybridoma lines in media free of serum. However, other proteins and macromolecules were always added to these media in order to allow long-term growth of the hybridoma lines. For example, serum-free media supplemented with serum proteins such as transferrin and insulin (Chang et al, J. Immunol. Methods, 39: 369 (1980)) or transferrin and insulin plus albumin containing liposomes (Andersson and Melchers, Curr. Top. Microbiol. Immunol., 81: 130 (1978)) have been used. Another medium used the dialyzable fraction of serum for small-scale cultivation with a two-chambered Marbrook vessel (Klinman and McKearn, J. Immunol. Methods, 42: 1 (1981)). Serum proteins were avoided in still another medium, but cultivation was restricted to 24-48 hours because of cell death (Galfre and Milstein, Methods in Enzymology, Vol. 73B, eds. Colowick and Kaplan, Academic Press, New York, page 1, 1981). However, none of these methods allows for the routine large-scale production of monoclonal antibodies in a protein-free culture medium, and such a production process and culture medium are still needed.
Several non-hybridoma mammalian cell types have been grown in either serum-free or protein-free media. For example, murine B lymphocytes have been shown to survive and grow in a medium containing insulin, transferrin and progesterone instead of serum (Mosier, J. Immunol., 127, 1490-1493 (1981)). A trace element mixture was used to replace serum in producing the protein-free medium MCDB 301, which is capable of supporting the growth of Chinese hamster ovary cell lines (Hamilton and Ham, In Vitro, 13, 537-549 (1977)). Such media, however, have not been applied to growing hybridoma cell lines or cells used for preparative production of biologically and clinically useful substances.